The blood-brain barrier (BBB) is one of the most restrictive barriers in biology. Numerous factors work together to create this restrictive barrier. Electron microscopy studies have demonstrated that tight junctions between brain vascular endothelial cells and other endothelial cell modifications (e.g., decreased pinocytosis, lack of intracellular fenestrate) prevented the formation of a plasma ultrafiltrate. Enzymatic activity at the BBB further limits entry of some substances, especially of monoamines and some small peptides (Baranczyk-Kuzma and Audus (1987) J. Cereb. Blood Flow Metab., 7:801-805; Hardebo and Owman (1990) Pathophysiology of the Blood-Brain Barrier, pp. 41-55 (Johansson et al., Eds.) Elsevier, Amsterdam; Miller et al. (1994) J. Cell. Physiol., 161:333-341; Brownson et al. (1994) J. Pharmacol. Exp. Ther., 270:675-680; Brownlees and Williams (1993) J. Neurochem., 60:793-803). Saturable, brain-to-blood efflux systems, such as p-glycoprotein (Pgp), also prevent the accumulation of small molecules and lipid soluble substances (Taylor, E. M. (2002) Clin. Pharmacokinet., 41:81-92; Schinkel et al. (1996) J. Clin. Invest., 97:2517-2524). Peripheral factors such as protein binding/soluble receptors, enzymatic degradation, clearance, and sequestration by tissues also affect the ability of a substance to cross the BBB by limiting presentation; these factors are especially important for exogenously administered substances (Banks and Kastin (1993) Proceedings of the International Symposium on Blood Binding and Drug Transfer, pp. 223-242 (Tillement et al., Eds.) Fort and Clair, Paris).
Substances are able to cross the BBB by way of a few pathways. Such pathways include: 1) saturable transporter systems, 2) adsorptive transcytosis wherein the compound to be transported is internalized by a cell in the BBB and routed to the abluminal surface for deposition into the brain intracellular fluid compartment, 3) transmembrane diffusion wherein the substance dissolves into the lipid bilayer which forms the membranes of the cells comprising the BBB, and 4) extracellular pathways wherein compounds exploit the residual leakiness of the BBB.
The hydrophilicity, the lack of stability due to enzymatic or chemical degradation, and the lack of transport carriers capable of shuttling polypeptides across cell membranes all play a part in precluding most polypeptides from transport into the brain. Several approaches to modify polypeptides to alter their BBB permeability have been attempted including: 1) conjugation with proteins that naturally cross the BBB (Raub and Audus (1990) J. Cell. Sci., 97:127-138; Banks and Broadwell (1994) J. Neurochem., 62:2404-2419; Bickel et al. (2001) Adv. Drug Deliv. Rev., 46:247-279); 2) modifying the polypeptide with cationic groups, i.e. “cationization” (Kumagai et al. (1987) J. Biol. Chem., 262:15214-15219; Triguero et al. (1989) Proc. Natl. Acad. Sci., 86:4761-4765; Triguero et al. (1991) J. Pharmacol. Exp. Ther., 258:186-192; Bickel et al. (2001) Adv. Drug Deliv. Rev., 46:247-279); 3) modifying the polypeptides with polyethylene glycol (PEG; Delgado et al. (1992) Crit. Rev. Ther. Drug. Carrier. Syst., 9:249-304; 4) linking the polypeptides to antibodies targeting certain cellular receptors (Zhang and Pardridge (2001) Brain Res., 889:49-56; Zhang and Pardridge (2001) Stroke, 32:1378-1384; Song et al. (2002) J. Pharmacol. Exp. Ther., 301:605-610) and 5) stearoylation of the polypeptide (Chekhonin et al. (1991) FEBS Lett., 287:149-152; Chopineau et al. (1998) J. Contr. Release, 56:231-237; Chekhonin et al. (1995) Neuroreport., 7:129-132).
Another method employed to increase BBB transport is by the use of Pluronic® block copolymers (BASF Corporation, Mount Olive, N.J.). Pluronic® block copolymers (listed in the US and British Pharmacopoeia under the name “poloxamers”) consist of ethylene oxide (EO) and propylene oxide (PO) segments arranged in a basic A-B-A structure: EOa—POb-EOa. This arrangement results in an amphiphilic copolymer, in which altering the number of EO units (a) and the number of PO units (b) can vary its size, hydrophilicity, and lipophilicity. A characteristic of Pluronic® copolymers is the ability to self-assemble into micelles in aqueous solutions. The noncovalent incorporation of drugs into the hydrophobic PO core of the Pluronic® micelle imparts to the drug increased solubility, increased metabolic stability, and increased circulation time (Kabanov and Alakhov (2002) Crit. Rev. Ther. Drug Carrier Syst., 19:1-72; Allen et al. (1999) Coll. Surfaces, B: Biointerfaces, 16:3-27).
Pluronic® micelles conjugated with antibody to alpha 2GP have been shown to deliver neuroleptic drugs and fluorescent dyes to the brain in mice (Kabanov et al. (1989) FEBS Lett., 258:343-345; Kabanov et al. (1992) J. Contr. Release, 22:141-157). Additionally, selected Pluronic® block copolymers, such as P85, are potent inhibitors of p-glycoprotein (Pgp) and increase entry of the Pgp-substrates to the brain across BBB (Batrakova et al. (1998) Pharm. Res., 15:1525-1532; Batrakova et al. (1999) Pharm. Res., 16:1366-1372; Batrakova et al. (2001) J. Pharmacol. Exp. Ther., 296:551-557). The mechanism of the Pluronic® effect in the latter case involves decreases in membrane microviscosity by Pluronic® and depletion of intracellular ATP, which together blocked the Pgp efflux function in brain endothelial cells (Batrakova et al. (2001) J. Pharmacol. Exp. Ther., 299:483-493; Batrakova et al. (2003) J. Pharmacol. Exp. Ther., 304:845-854). Pluronic® did not induce toxic effects in the BBB as revealed by the lack of alteration in paracellular permeability of the barrier (Batrakova et al. (1998) Pharm. Res., 15:1525-1532; Batrakova et al. (2001) J. Pharmacol. Exp. Ther., 296:551-557). It has been noted that Pluronic® fluorescently labeled with FITC can accumulate in the brain following systemic administration in mice (Kabanov et al. (1992) J. Contr. Release, 22:141-157). Furthermore, selected Pluronic® copolymers can cross the membranes of the brain endothelial cells (Batrakova et al. (2003) J. Pharmacol. Exp. Ther., 304:845-854).
Notably, Pluronic® copolymers have also been used in combination with anticancer drugs in the treatment of multidrug resistant (MDR) cancers (Alakhov et al. (1996) Bioconjug. Chem., 7:209-216; Alakhov et al. (1999) Colloids Surf., B: Biointerfaces, 16:113-134; Venne et al. (1996) Cancer Res., 56:3626-3629). Indeed, Phase I and II clinical trial are being performed on doxorubicin formulated with Pluronic® (“SP1049C”) for the treatment of adenocarinoma of esophagus and soft tissue sarcoma, both cancers with high incidence of MDR (Ranson et al. (2002) 5th international symposium on polymer therapeutics: from laboratory to clinical practice, pp. 15, The Welsh School of Pharmacy, Cardiff University, Cardiff, UK).